Recent population expansion and increased migration linked to urbanization are assumed to be eroding the genetic structure of human populations. We investigated change in population structure over three generations by analysing both demographic and mitochondrial DNA (mtDNA) data from a random sample of 2351 men from 22 Iranian populations. Potential changes in genetic diversity () and genetic distance (FST) over the last three generations were analysed by assigning mtDNA sequences to populations based on the individual's place of birth or that of their mother or grandmother. Despite the fact that several areas included cities of over one million inhabitants, we detected no change in genetic diversity, and only a small decrease in population structure, except in the capital city (Tehran), which was characterized by massive immigration, increased and a large decrease in FST over time. Our results suggest that recent erosion of human population structure might not be as important as previously thought, except in some large conurbations, and this clearly has important implications for future sampling strategies.